Protein extraction methods

Jiang X, Jiang X, Feng S, et al. Development of efficient protein extraction methods for shotgun proteome analysis of formalin-fixed tissues. J. Proteome Res. 2007 6 1038-1047. 

Protein extraction methods are critical. In previous studies various extraction procedures have been tested. Paris et al. [20] could show that after breaking A. alternata cells, the use of carbonate buffer supplemented with protease inhibitors (phenylmethylsulfonyl fluoride) and phenol-binding components... 

Our knowledge of leaf protein can be traced back over 226 years (Rouelle, 1773) [5]. The method of processing-preserving leaf protein suitable for human consumption was first patented in 1927 by Ereky [6], and a quarter of a century later some widespread experiments were carried out in the field in England [5]. An entirely different, novel procedure, the VEPEX (Vegetable Protein Extract) method was elaborated and the first leaf protein plant in the world was set up in Hungary in 1972 [7] (Fig. 2). 

Similar to intact cell-based methods, several protocols have been described to extract proteins from cells. The most cormnonly used protein extraction method is an ethanol-formic extraction, in which a erode protein extraction is performed in a microcentrifuge tube (e.g., Freiwald and Sauer 2009). In addition, plate-based formic acid extraction has also been described (e.g., Schirlthess et al. 2014). 

Extraction of lyophilized protein powder by w/o-ME solution (solid-phase extraction method, SPE). 

Unlu, M., Morgan, M.E., Minden, J.S. (1997). Difference gel electrophoresis a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071-2077. 

NA isolation and molecular characterization will be important to define the origin and functions of these proteins. At this time, infected cell nuclei offer the only source of these proteins, and NA have proved resistant to classic nuclear extraction methods (Yao and Jasmer, 1998). NA can be solubilized under conditions that co-extract nuclear lamins a/c and b (4 M urea, pH 8.0). Despite these similar physical properties, NA do not co-localize with lamins in the nucleoskeleton. However, both disulphide bonds and ionic interactions appear to contribute to nuclear complexes containing NA. In addition, NA can be cross-linked within host nuclei with protein cross-linking reagents. The foregoing properties represent current information available for the development of strategies to isolate and characterize these proteins and to investigate host proteins with which NA interact. 

Many diseases are characterized by the expression of specific proteins1 in some cases, malignant cells yield unique protein profiles when total cellular protein extracts are analyzed by proteomic methods such as two-dimensional gel electrophoresis or matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS).2 High-throughput proteomic studies may be useful to differentiate normal cells from cancer cells, to identify and define the use of biomarkers for specific cancers, and to characterize the clinical course of disease. Proteomics can also be used to isolate and characterize potential drug targets and to evaluate the efficacy of treatments. 

In summary, studies carried out with tissue surrogates25 highlight some of the problems that must be overcome before proteins extracted from FFPE tissues can be used for routine proteomic studies. First, these studies demonstrate that reversal of protein-formaldehyde adducts does not assure quantitative extraction of proteins from FFPE tissues or vice-versa. It may ultimately turn out that there is no one universal method that can accomplish both tasks, but that instead, each step will need to be optimized separately. Studies with tissue surrogates also suggest that failure to quantitatively extract the entire protein component from FFPE tissues may result in sampling bias due to the preferential extraction of certain proteins. This behavior may be linked to protein physical properties, such as the isoelectric point. The results of our... 

High-throughput proteomic methods hold great promise for the discovery of novel protein biomarkers that can be translated into practical interventions for the diagnosis, treatment, and prevention of disease. These approaches may also facilitate the development of therapeutic agents that are targeted to specific molecular alterations in diseases such as cancer. In many cases, malignant cells yield unique protein profiles when total protein extracts from such cells are analyzed by 2-D gel electrophoresis or mass spectrometry (MS) methods. Such proteomic studies have the potential to provide an important complement to the analysis of DNA and mRNA extracts from these tissues.1... 

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